We developed a LC-MS-MS assay of the (2)H labeling of free glutathione (GSH) and bound glutathione [GSSR; which includes all DTT-reducible forms, primarily glutathione disulfide (GSSG) and mixed disulfides with proteins] and ophthalmate (an index of GSH depletion) labeled from (2)H-enriched body water. In rats whose body water was 2.5% (2)H enriched for up to 31 days, GSH labeling follows a complex pattern because of different rates of labeling of its constitutive amino acids. In rats infused with [(13)C(2),(15)N-glycine]glutathione, the rate of appearance of plasma GSH was 2.1 micromol.min(-1).kg(-1), and the half-life of plasma GSH/GSSR was 6-8 min. In healthy humans whose body fluids were 0.5% (2)H enriched, the (2)H labeling of GSH/GSSR and ophthalmate can be precisely measured after 4 h, with GSH being more rapidly labeled than GSSR. Since plasma GSH/GSSR derives mostly from liver, this technique opens the way to 2) probe noninvasively the labeling pattern and redox status of the liver GSH system in humans and 2) assess the usefulness of ophthalmate as an index of GSH depletion.